The type I interferons (IFNa and IFN[unreadable]) are potent regulators of cell growth with inhibitory effects against many human cancers. However, their clinical use has been limited by inability to achieve effective concentrations of IFN at sites of tumor without systemic toxicity. Our goal is to overcome this limitation using the tumor-targeting ability of monoclonal antibodies to carry IFNs directly to cancer sites. We hypothesize that antibody-IFN fusion proteins could be highly effective cancer therapeutic agents, selectively localizing IFN to sites of tumor. We have recently succeeded in producing fusion proteins targeting the CD20 antigen expressed on the surface of B cell non-Hodgkin lymphomas. Anti-CD20-IFNa fusion proteins containing murine or human IFNa exhibited potent anti-tumor effects against a human CD20+ murine lymphoma and human B cell lymphoma both in vitro and in vivo. We now propose to further characterize and optimize these IFN fusion proteins and to extend our studies to include fusion with IFN[unreadable], which is up to 10-fold more potent than IFNa. To achieve this, we propose the following Specific Aims: Aim 1: Characterize the efficacy of anti-CD20 fused with murine IFNa (mIFNa) or murine IFN[unreadable] (mIFN[unreadable]) against mouse lymphomas in vitro and in vivo and determine their mechanisms of action. A. Characterize the in vitro activity of fusion proteins against two murine B cell lymphoma cell lines engineered to express human CD20, including their ability to inhibit tumor cell proliferation, induce apoptosis, and mediate antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cellular cytotoxicity (CDC). B. Characterize the in vivo efficacy of anti-CD20-mIFNa and anti-CD20-mIFN[unreadable] against 38C13-huCD20 in C3H mice and A20-huCD20 in BALB/c huCD20-transgenic mice. Aim 2: Evaluate the efficacy of anti-CD20 fused with human IFNa (hIFNa) against human lymphomas in vitro and in vivo and determine the mechanism(s) of action. A. Using human B cell lymphoma cell lines representing various histologies, characterize the in vitro activity of anti-CD20-hIFNa as in Aim 1. B. Evaluate the in vivo efficacy of anti-CD20-hIFNa against human B cell lymphoma xenografts in SCID mice. C. Measure the activity of anti-CD20-hIFNa against primary human B cell lymphoma specimens in vitro. Aim 3: Construct anti-CD20 fusions with human IFN[unreadable] in an attempt to increase anti-tumor potency. A. Explore the use of alternative peptide linkers to optimize the stability and IFN activity of the fusion protein. B. Characterize the most promising anti-CD20-hIFN[unreadable] fusion proteins. Aim 4: Explore the possibility of further enhancing anti-CD20-hIFNa/[unreadable] efficacy using clinically-relevant pharmacologic inhibitors of lymphoma cell survival pathways. A. Evaluate the in vitro efficacy of the fusion proteins against a panel of lymphoma cell lines and primary cells when used in combination with inhibitors of mTOR (rapamycin or temsirolimus) or PI3 kinase d (CAL-101). B. Evaluate the in vivo efficacy of the fusion proteins against human lymphoma xenografts in combination with inhibitors of mTOR (temsirolimus) or PI3 kinase d (CAL-120). PUBLIC HEALTH RELEVANCE: Lymphoma remains a major health problem with over 66,000 individuals in the United States succumbing each year from the disease. In this research effort we will further develop and characterize novel therapeutic proteins that show great promise for the treatment of lymphoma even in cases where the lymphoma is resistant to current therapies. Importantly the technology that we are developing is not limited to the treatment of lymphoma, but can be applied to the treatment of many different cancers including melanoma, prostate, renal cell carcinoma and multiple myeloma.